Use of control areas to detect interfering samples in a detection method

ABSTRACT

A solid phase with at least one test area is described which contains reagents for the detection of at least one analyte in a sample, wherein the solid phase additionally comprises at least one control area for the detection of interfering reactions.

[0001] The invention concerns a solid phase with at least one test areato detect an analyte which additionally comprises at least one controlarea to detect interferences. Furthermore the invention concerns amethod for the detection of one or several analytes using a solid phaseaccording to the invention in which interfering reactions can bedetected and corrected if necessary.

[0002] When analytes are detected by binding assays interferences in thedetection reaction occur with some samples which lead to false testresults. This phenomenon is usually referred to as a matrix effect ofthe sample. The presence of an interference cannot in general beindicated in conventional test formats. It is attempted to preventmatrix effects of the various samples or to reduce them as far aspossible by elaborate optimization of the solid phase, test buffers anddetection reagent. However, such a reduction of interference indetection methods is complicated and expensive. Moreover one cannotcompletely rule out the possibility that an adequate reduction ofinterference does not occur for certain samples despite testoptimization since it is unfortunately not possible to completelysuppress matrix effects. In addition new interferences which wereunknown during the development of the detection method can occur whichalso lead to false results. Consequently there is a problem that in theknown detection methods false test results can be obtained without theuser being aware of this. This circumstance is particularly tragic inthe case of qualitative tests for the detection of an infectiousdisease. Thus for example a false positive sample resulting from matrixproblems has considerable consequences for a HIV test.

[0003] U.S. Pat. No. 4,558,013 describes a test strip which contains anon-defined uncoated negative control region in addition to test regionscoated with specific test reagents. The value measured in the testregion is corrected by subtracting the unspecific binding in the controlregion. However, such a procedure can only partially correct forinterferences since the unspecific binding of interfering components tothe test region usually differs considerably from the binding ofinterfering components to the uncoated control region.

[0004] U.S. Pat. No. 5,356,785 describes a solid phase with several testareas which each contain different amounts of a solid phase receptor forthe detection of an analyte. The solid phase additionally contains areference area which generates a detectable signal of known intensitywith the test reagent. Control areas to determine unspecificinteractions between the sample and the solid phase are not disclosed.

[0005] U.S. Pat. No. 4,916,056 (Brown III et al.) describes a solidphase for the qualitative or quantitative determination of an analyte,in particular of an antigen, antibody or DNA segment in a sample. Thesolid phase contains, in addition to a test area, a reference area whichyields a positive signal in the test and a non-defined negative controlarea which contains the positive reference area and the test area. Adisadvantage of this device is that the surface of the uncoated controlregion differs too greatly from the test area to achieve an effectivecorrection for interferences.

[0006] Hence an object of the invention was to provide devices andmethods for the detection of analytes which enable a direct indicationof the presence and optionally the type of interferences such that theseinterferences can be taken into account when evaluating the testresults.

[0007] This object is achieved according to the invention by a solidphase having at least one defined test area for the detection of ananalyte in a sample which is characterized in that the solid phaseadditionally comprises at least one defined control area for thedetection of interferences. In this connection the term “defined testareas” on a solid phase is understood to mean that the test areascomprise defined regions of the solid phase which are preferablyspatially separated from other test areas by inert regions. The definedtest areas preferably have a diameter of 10 μm to 1 cm and particularlypreferably 10 μm to 5 mm. Miniaturized test areas with a diameter of 10μm to 2 mm are most preferred. Solid phases with several test areas arepreferred which are also referred to as array systems. Such arraysystems are for example described in Ekins and Chu (Clin. Chem. 37(1995), 1955-1967) and in U.S. Pat. Nos. 5,432,099, 5,516,635 and5,126,276. An advantage of array systems is that several analyte andcontrol determinations can be carried out simultaneously on one sample.The use of control areas to detect unspecific binding and/or interferingsamples can considerably improve the reliability of the resultsespecially with miniaturized array test systems.

[0008] In this connection the detection of interferences and unspecificbinding in qualitative tests and in particular in those which havestringent requirements for specificity such as tests for infections(e.g. HIV) are of particular interest. The indication of an interferenceand correction of the test result enables false-positive results to beconsiderably reduced thus leading to an enormous improvement ofspecificity.

[0009] The solid phase according to the invention is any conventionalsupport for detection methods, preferably a non-porous support e.g. asupport with a plastic, glass, metal or metal oxide surface. Poroussupports such as test strips are also suitable. Spatially discreteregions (test areas) are located on this support. Immobilized solidphase receptors are applied to these test areas. The solid phasereceptors are immobilized by known methods e.g. by direct adsorptivebinding, by covalent coupling or by coupling via high affinity bindingpairs e.g. streptavidin/biotin, antigen/antibody or sugar/lectin. Thepresence or/and the amount of the analyte in a sample can be determinedby specific binding of components from the detection medium e.g. of theanalyte to be determined or of an analyte analogue to the solid phasereceptor.

[0010] The detection of the analyte and the presence of interferingreactions is achieved in the method according to the invention in aknown manner by using suitable marker groups e.g. fluorescent markergroups. Alternatively with suitable solid phases it is possible to alsodetect the interaction of components of the detection medium with thetest and control areas by determining the layer thickness of therespective area e.g. by plasmon resonance spectroscopy.

[0011] With array systems in which several analytes from a sample aredetected simultaneously, it is preferable to use a “universal” markergroup which enables a simultaneous detection of several differentanalytes to different test areas. An example of such universal markergroups are marker groups which carry a receptor that can specificallyinteract (e.g. by means of a high-affinity binding pair such asantibody/antigen or streptavidin/biotin etc.) with a complementaryreceptor on a test reagent e.g. a soluble receptor for an analyte to bedetermined or for an analyte analogue.

[0012] The application of such a universal marker group is exemplifiedin FIG. 1. In this case a fluorescent latex bead which is coupled withan anti-digoxigenin antibody (<Dig> label) is used for three differenttest formats on a single solid phase i.e. to determine HIV antibodies,HBs antigen and anti-HBc antibodies. In the case of the anti-HIVantibody determination an immobilized HIV antigen and a digoxigenylatedsoluble HIV antigen are used which form an immobilized immune complexwith the anti-HIV antibodies to be detected. The marker group can bindto the digoxigenin groups present on this immune complex. For thedetermination of HBs antigen, an immobilized antibody and adigoxigenylated soluble antibody that can interact with the marker groupare used in a corresponding manner. A competitive test format is usedfor the anti-HBc determination in which anti-HBc antibodies present inthe sample compete with a digoxigenylated anti-HBc antibody forimmobilized HBc antigen. The quantity of marker group bound to the testarea is inversely proportional to the anti-HBc concentration in thesample.

[0013] Defined test and control areas can additionally contain adetectable and analyte-unspecific marker group which can be detectedconcurrently with the analyte-specific marker group and does notinterfere with it, in order to differentiate them from inert regions ofthe solid phase. An example of such an analyte-unspecific marker groupis a fluorescent marker group which fluoresces at a wavelength which isdifferent from the fluorescent wavelength of an analyte-specific markergroup. The analyte-unspecific marker group is preferablyimmobilized—like the solid phase receptor—via a high affinity bindingpair e.g. streptavidin/biotin.

[0014] The solid phase according to the invention can be used in anydetection methods e.g. in immunoassays, nucleic acid hybridizationassays, sugar-lectin assays and similar methods.

[0015] The solid phase according to the invention enables a detection ofinterfering reactions to obtain reliable test results even when themeasures known from the prior art for interference reduction are notadequate for certain samples. The control areas not only enable aqualitative detection of interfering reactions but also in many casesenable a quantitative correction for the interference.

[0016] The solid phase can comprise several, in particular differentcontrol areas for the detection of interfering reactions in order todetect different interferences. In this manner it is possible tospecifically detect different types of interference. It is particularlyadvisable to apply a series of control areas that are suitable for thedetection of frequently occurring or/and particularly relevantinterfering components for the respective test.

[0017] Interference of test procedures can in general be the result ofundesired, non-analyte-specific interactions of substances on the testarea with components of the detection medium. These substances on thetest area may be in particular components of the solid phase, parts ofthe solid phase receptor and other reagents located on the surface ofthe solid phase. The interfering components of the detection mediummainly come from the sample (matrix effect) and in some cases lead tounspecific binding of test reagents e.g. of the detection reagent to thesolid phase and result in a falsification of the measured signal. Thusinterfering interactions between the test area and sample components,test reagents, reaction products or complexes of sample components andtest reagents may occur.

[0018] The solid phase according to the invention preferably comprisescontrol areas to detect interferences that are due to an undesiredbinding of components of the detection medium to the specific solidphase receptor for the analyte. Non-analyte interfering components arefrequently present in samples such as antibodies or antigens which havea tendency for an increased unspecific binding to the solid phasereceptor and in this manner lead to erroneous test results. Hence acontrol area is particularly advantageous which comprises a non-analytespecific solid phase receptor which, with the exception of the regionthat can specifically bind to the analyte, is completely identical tothe solid phase receptor in the test areas.

[0019] If the solid phase receptor is for example an antibody or anantibody fragment, a control area is used which contains an unspecificantibody or an unspecific antibody fragment of the same species,preferably of the same class and particularly preferably of the samesubclass as that of the solid phase receptor of the test area. If thesolid phase receptor is for example an antigen e.g. a peptide or apolypeptide, a control area is used which contains a mutated “antigen”which differs from an immunologically reactive antigen by modificationse.g. by modifying the smallest possible number of amino acids in theregion of immunogenic epitopes. These amino acid modifications cancomprise insertions, deletions and preferably substitutions of naturalamino acids by other natural amino acids or non-natural amino acidderivatives e.g. D-amino acids. If the solid phase receptor is forexample a nucleic acid, a control area is used which contains a“mutated” nucleic acid which differs from the nucleic acid immobilizedon the test area by modifications in the nucleotide sequence for exampleby a base substitution within the sequence responsible for therecognition of a target nucleic acid, preferably in the middle thereof.A mutein of the solid phase receptor is most preferably used which, withthe exception of the analyte-specific antigen binding site, iscompletely identical to the solid phase antibody in the test areas.

[0020] It is also preferable that the solid phase receptor applied tothe control area has been subjected to identical treatment steps e.g.derivatizations as the solid phase receptor applied to the test area.Thus for example—in the case of a biotinylated solid phase receptor—thenumber of biotin molecules coupled to the solid phase receptor should bethe same in the test area and in the control area. In addition the solidphase receptors in the test area and in the control area should havebeen subjected to an identical coupling chemistry. In addition identicallinkers should also have been used. A solid phase receptor suitable fora control area should not be able to specifically bind to the analytebut preferably comprises all other regions and thus binding sites of thesolid phase receptor of the test area. Consequently the unspecificbinding of interfering components to the respective solid phase receptorof the test area and control area is essentially identical so that it ispossible to quantitatively correct the measured value of the test areaon the basis of the measured value of the control area.

[0021] The solid phase additionally comprises at least one control areato detect interferences which are caused by the reaction of otherimmobilized reagents in the test areas with non-analyte components ofthe sample. As a result interfering components are especially detectedthat react specifically or/and unspecifically with components of theloading solution used to apply the solid phase receptor. Such a controlarea can for example contain reagents of the loading solution used in atest system such as buffers, immobilization reagents such asstreptavidin, biotinylated substances e.g. biotinylated fluorescentmarkers, non-analyte specific antibodies etc., blocking reagents orlinkers.

[0022] Rheumatoid factors often interfere with tests. Hence it ispreferable to set up at least one control area on the solid phase todetect rheumatoid factors. Rheumatoid factors are usually IgM moleculesbut rarely also IgG, IgA and IgE molecules which react with the Fc partof antibodies and interfere with the test if they for examplecross-react with the antibody immobilized on the test area or/and asoluble detection antibody, e.g. by cross linking the antibody bound tothe solid phase with a labelled detection antibody which results in anunspecifically increased signal. For such a control area an unspecificIgG molecule, preferably the Fcy part of a human IgG molecule, isapplied to the solid phase. Then the rheumatoid factor does not onlybind to the test area during the test but also to the control area andthus indicates the interference.

[0023] A further interference which occurs relatively frequently iscaused by foreign-species-specific antibodies in the sample i.e.antibodies which are directed against antibodies of foreign species e.g.human anti-mouse antibodies (HAMA). Thus the solid phase according tothe invention also preferably comprises at least one control area totest for foreign-species-specific antibodies. Foreign-species-specificinterfering components e.g. RAMA lead for example in a double antibodysandwich assay to a cross linking of the solid phase antibody with thedetection antibody and consequently to an unspecifically increasedsignal. An unspecific antibody of the same species as the test antibodyis preferably used for a suitable control area. A HAMA interferingcomponent which may be present in the sample binds to the antibodyapplied to the control area which indicates the interfering reaction.

[0024] Instead of a solid phase receptor that is identical with thedetection reagent in the test area apart from the specificanalyte-bindable region, the complex formed during the detectionreaction e.g. solid phase antibody-analyte-detection antibody (withoutlabel) can also be applied to a control area so that a specific reactionis no longer possible although the unspecific binding sites are almostidentical with those of the test area.

[0025] Interfering components in the serum are also known which aredirected against neo-epitopes of an antibody fragment. Such neo-epitopesare formed for example during the F(ab′)₂ cleavage of an IgG molecule.In this case it is preferable to provide a control area which contains afragment of an unspecific antibody which has been produced by the samemethod (cleavage conditions and cleavage protein) as the antibodyfragments of the test area. The interfering components bind to theseunspecific antibody fragments and can therefore be detected.

[0026] In addition it is also possible using suitable control areas todetect interfering components which may be present in a sample e.g.interfering antibodies that are directed against immobilization reagentson the test areas such as streptavidin. For this purpose streptavidin(SA) is applied to a control area and labelled SA is added to thedetection reagent. If anti-SA antibodies are present in the sample, asandwich complex is formed and the antibody is specifically detected.

[0027] The solid phase according to the invention can further contain acontrol area to detect the total IgE content. Such a control area isparticularly recommended for allergy tests. When specific IgEs aredetermined in allergy diagnostics samples with a high total IgE contentoften interfere with the specific detection reaction of a certain IgE.The total IgE content can be determined in parallel to the detectionmethod with the aid of a control area on which anti-IgE antibodies havebeen applied.

[0028] Finally control areas can also be provided to detectinterferences that are caused by reactions of components of thedetection medium with the solid phase support. For this purpose allcomponents of the solid phase support such as the support material (e.g.polystyrene), plasticizer, functional groups etc. can be applied to acontrol area.

[0029] In addition control areas to determine a cut-off value can beused for certain test procedures e.g. in qualitative orsemi-quantitative tests for infectious diseases, allergies etc. The“cut-off” value is a threshold value that is set for test procedures inorder to differentiate between positive and negative values. Such a“cut-off” value is of particular importance for test procedures whichrelate to infectious diseases. One possibility is to use a “negative”control area which can contain a loading solution without the testreagent or a mutein of the test reagent. A major advantage is that asample-specific value for the unspecific binding is determined for eachsample and thus an improved specificity of the test is obtained. In thismanner a separate negative control can be omitted.

[0030]FIG. 2 shows the effect of a control area used in a test in whicha cut-off value is employed. With a large number of samples a certainnumber of false-negative and false-positive results are obtained (leftside) with a conventional test procedure. The use of control areasaccording to the invention (right side) can selectively reduce thesignals from negative samples which leads to a reduction of the cut-offvalue. In this way a positive/negative differentiation can be made witha considerably reduced probability of error.

[0031] A positive control can also be simulated by a reference areawhich contains the analyte or an analyte-like reagent. A major advantageis that a combination of a negative control area with a positivereference area enables a sample-specific cut-off value to be determinedfor each individual measurement. An advantage of this is that anindirect calibration is not necessary and an improved test specificityis achieved.

[0032] It is of course possible to provide other or/and additionalcontrol areas in addition to the aforementioned preferred control areasdepending on the test procedure in order to detect interferingcomponents that are presumed to be or are present in a sample.

[0033] The control areas provided according to the invention not onlyenable a qualitative detection of interfering reactions but often also aquantitative correction for the interference. With a suitable selectionof test conditions the unspecific binding of non-analyte componentsoccurring in the test areas is accurately reflected in certain controlareas. Thus the measured value in a test area can be simply corrected toobtain a correct and unbiased result. Even if the unspecific binding inthe test area and the control area are not identical, the measuredsignal can be corrected in qualitative tests since in this case only astatement of “positive” or “negative” is necessary.

[0034] An additional advantage is that the solid phase according to theinvention enables the presence of several interfering components to bedetected separately and it is possible to determine the type ofinterference. The user recognizes immediately that the result may befalsified due to the presence of one or several interfering components.The user can then either correct the measured results on the basis ofthe data that were determined, repeat the measurement with another testformat or pretreat the sample in a suitable manner e.g. by separatingthe interfering components.

[0035] The solid phase according to the invention can be used to detectan analyte in a sample e.g. in a body fluid such as blood, serum,plasma, saliva etc. Solid phases with several test areas, i.e. arraysystems, for the simultaneous detection of several analytes arepreferably used. At least one control area is preferably used for eachtest area in such array systems. The solid phases according to theinvention can be used in all known heterogeneous test proceduresespecially in immunoassays and nucleic acid hybridization assays.

[0036] Hence a further subject matter of the invention is a method forthe detection of an analyte using a solid phase with at least onedefined test area which in addition comprises at least one control areato detect interfering reactions. The method according to the inventionpreferably includes the use of the control areas for the quantitativecorrection of interferences.

[0037] Finally the invention also concerns the use of control areas in amethod for the detection of an analyte for the simultaneous detection ofinterferences.

[0038] The invention is further elucidated by the following figures andexamples.

[0039]FIG. 1: shows an example of a miniaturized array system(microspot) that uses a universal marker group and allows thesimultaneous determination of 3 parameters on a solid phase;

[0040]FIG. 2: shows the specificity improvement that is achieved by theuse according to the invention of control areas in test formats in whicha cut-off value is determined and

[0041]FIG. 3: shows a schematic representation of a solid phaseaccording to the invention for the determination of HBs antigen thatcontains several control areas (<CK-MB>; <TNT> and <TSH>) in addition tothe test area (<HBsAg>).

EXAMPLES Example 1 HBsAg Test Procedure

[0042] A monoclonal antibody to HBsAg is applied to a test area of ca.100 μm on a polystyrene support. A multiple determination can be carriedout without additional work when one test is carried out per samplepipetting by applying an identical reagent solution several times. 30 μlsample prediluted with sample buffer is pipetted onto the test area andincubated for 20 minutes at room temperature while shaking. Afteraspirating the sample and washing the test field with wash buffer, 30 μlreagent solution 1 containing digoxigenin(Dig)-labelled anti-HBsAgantibody is added by pipette and again incubated for 20 minutes at roomtemperature while shaking. After aspirating reagent solution 1 andwashing the test field with wash buffer, 30 Al reagent solution 2containing the detection reagent is pipetted onto the test field. 100 nmfluorescent dyed latex particles which are covalently coated with ananti-Dig antibody are used as a detection reagent. This detectionreagent is in turn incubated for 20 minutes at room temperature whileshaking, subsequently aspirated, washed and sucked dry. The test fieldis irradiated with a He—Ne laser at 633 nm wavelength and thefluorescence at 670 nm wavelength is measured with a CCD camera. Aschematic representation of this test format is shown in the middle ofFIG. 1.

[0043] The following test-specific reagents were used:

[0044] Solid phase antibody: monoclonal mouse anti-HBsAg antibody 1(Fab′₂ fragment) biotin conjugate 1:1 subtype IgG1

[0045] Detection antibody: monoclonal mouse anti-HBsAg antibody 2IgG-Dig conjugate 1:10

[0046] The following test results (counts) were measured: negative con-signal test signal test trol field* field negative cut-off Sample(counts) (counts) control field index** negative control 22 22 0 0.0positive contr. 1 20 5459 5439 82.4 (20 U/ml) positive contr. 2 27 760733 11.1 (3 U/ml) negative sample 1 15 15 0 0.0 negative sample 2 15 150 0.0 negative sample 3 14 14 0 0.0 negative sample 4 15 15 0 0.0negative sample 5 15 15 0 0.0 negative sample 6 17 17 0 0.0

[0047] The test is positive at a cut-off >1. The test is negative at acut-off <1.

Example 2 HBsAg Test With Control Spots to Detect HAMA Interferences

[0048] Additional unspecific monoclonal antibodies (MABs) were appliedin addition to HBsAg-specific antibodies to detect HAMA interference(see FIG. 3). Antibodies to creatinine kinase MB (<CK-MB>), Troponin(<TNT>) and thyroid-stimulating hormone (<TSH>) were used in the form ofFab′₂ or Fab′ biotin conjugates. Due to the fact that the humananti-mouse antibodies in the sample cross-link the unspecific MABs boundto the solid phase and the specific detection antibody it is possible todetect the so-called HAMA interference. The aim of the experiment was tofind an optimal MAB for HAMA detection. The HBsAg/control panel wasmeasured with 6 different HAMA samples. For this purpose no or 100 μg/mlHAMA interference-reducing reagent were added to the sample buffer.

[0049] a) Experimental results without HAMA interference-reducingreagent MAB<HBsAg> MAB<TSH> MAB<TNT> MAB<CK-MB> cut-off F(ab′)₂-Bi 1:1*F(ab′)₂-Bi 1:1* Fab′-Bi 1:1* Fab′-Bi 1:1* index** Sample (counts)(counts) (counts) (counts) HBsAg neg. contr. 3 18 0 10 0.04 pos. contr.6169 23 0 5 73.4 (20 U/ml) HAMA 1 1598 2808 741 177 19.0 HAMA 2 488910434 306 42 58.2 HAMA 3 737 3728 29 32 8.8 HAMA 4 358 2513 0 0 4.3 HAMA5 43 2565 0 0 0.5 HAMA 6 32157 32108 32717 32930 382.8

[0050] This result shows that 5 of the 6 HAMA samples interfere stronglywith the HBsAg test and lead to a false-positive result. Theinterference can be unambiguously indicated by the presence of thecontrol spots. The TSH-MAB which has an identical cleavage fragment(Fab′₂), an identical biotin stoichiometry and identical couplingchemistry is most suitable for this. In contrast the two MABs with Fab′fragments react more poorly than the HBsAg-MAB with the HAMA interferingsamples and are therefore less suitable for identifying all HAMAinterferences.

[0051] b) Experimental results with 100 μg/ml HAMA interference-reducingreagent MAB MAB <HBsAg> <TSH> F(ab′)₂- F(ab′)₂- MAB<TNT> cut-off cut-offBi 1:1* Bi 1:1* Fab′-Bi 1:1* index** index Sample (counts) (counts)(counts) HBsAg HBsAg_(corr.)*** neg. contr. 0 0 0 0.0 0.0 pos. contr.4186 0 0 41.0 41.0 (20 U/ml) HAMA 1 0 0 0 0.0 0.0 HAMA 2 0 0 0 0.0 0.0HAMA 3 0 0 0 0.0 0.0 HAMA 4 0 0 0 0.0 0.0 HAMA 5 0 0 0 0.0 0.0 HAMA 6144 187 217 1.4 0.0

[0052] The addition of a very large amount of 100 μg/mlinterfering-reducing reagent which would make the test too expensive forroutine application enables an interference elimination in 5 of the 6HAMA samples. However, the HAMA sample 6 remains a problem whoseinterference cannot be reduced despite the high concentration ofinterference-reducing reagent. The two control spots can be used toclearly indicate the HAMA interference that is still present. Since theunspecific binding in the TSH control spot corresponds to the unspecificbinding in the specific HBsAg spot, the specific signal can be correctedwith the aid of the signal in the control spot. With this measure thissample also becomes clearly negative which leads to a considerableimprovement of the specificity. Hence the concentration ofinterference-reducing protein in the sample buffer and thus theproduction costs can be considerably reduced with the aid of the controlspots to improve specificity.

Example 3 HBsAg Test with Control Spots to Detect Sample-specific MatrixEffects

[0053] It is generally known that unspecific binding of the detectionreagent can be caused by various matrix effects. The application ofcontrol spots which have a comparable behaviour towards matrix effectsas the test-specific spot enables such matrix effects to be indicatedand optionally even to be corrected. This consequently leads to aconsiderable improvement of specificity.

[0054] In the following experiment a HBsAg test was again measured usingvarious HBsAg-negative samples which were selected on the bases ofmarked matrix effects. MAB <HBsAg> cut-off F(ab′)₂- MAB<TSH> cut-offindex Bi 1:1* F(ab′)₂-Bi 1:1* index** HBsAg_(corr) Sample (counts)(counts) HBsAg *** neg. control 3 18 0.04 0.0 pos. control 6169 23 73.473.2 (20 U/ml) negative sample 3415 0 0 0.0 0.0 negative sample 3418 65102 0.77 0.0 negative sample 4561 128 494 1.52 0.0 negative sample 4567158 328 1.88 0.0 negative sample 4609 0 0 0.0 0.0 negative sample S21 2552 0.30 0.0 negative sample S25 4 3 0.05 0.01 negative sample S45 16 300.19 0.0 negative sample S49 0 0 0.0 0.0

[0055] Of the measured 9 conspicuously negative samples, 2 samples leadto false-positive results. All samples were brought to zero bycorrecting the measured value with the aid of a control spot and arethus unequivocally negative.

Example 4 HBsAg Test with Control Areas to Detect Interferences byRheumatoid Factors

[0056] Additional MABs of the IgG1 subtype were applied in addition tothe HBsAg-specific antibody in order to detect interference byrheumatoid factors. Since rheumatoid factors can react with the Fc partof antibodies, this can cause a cross-linking of the test-specific MABsbound to the solid phase and the specific detection antibody and thusgenerate a false-positive reaction. The aim of this experiment was tofind the optimal MAB to detect rheumatoid factors. For this reasondifferent MABs of the IgG1 subclass were applied. This HBsAg/controlpanel was measured with a negative control, 3 different positivecontrols, 4 normal negative samples and 5 negative samples withincreasing amounts of rheumatoid factors (RF 1-5). The respectivecontent of HBsAg in the positive controls and the content of rheumatoidfactors in the rheumatoid factor samples is stated in U/ml. The resultswere as follows: MAB negative <HBsAG> MAB<IgE> MAB<IgE> MAB<IgE> controlcut-off IgG-Bi “1”IgG-Bi “2”IgG-Bi “3”IgG-Bi field* index* Sample[counts] [counts] [counts] [counts] [counts] HBsAg neg contr 27 34 31 3027 0.0 pos contr 4701 44 44 44 44 57.5 (20 U/ml) pos contr 842 43 43 4343 9.9 (5 U/ml) pos contr 106 31 31 31 31 0.9 (0.1 U/ml) neg sample 1 3035 30 30 30 0.0 neg sample 2 28 33 33 34 28 0.0 neg sample 3 29 35 29 2929 0.0 neg sample 4 27 32 27 27 27 0.0 RF 1 39 34 29 30 27 0.15 (307U/ml) RF 2 41 33 28 28 28 0.16 (421 U/ml) RF 3 429 33 115 64 30 4.9(1307 U/ml) RF 4 5790 47 865 204 47 70.9 (1793 U/ml) RF 5 7530 106 2527759 54 92.3 (2599 U/ml)

[0057] This example clearly shows that negative samples with aconcentration of rheumatoid factors >1000 U/ml considerably interferewith the HBsAg test and lead to a false-positive result. 2 of the 3tested MAB control areas simulate the interference of the HBsAg test andalso yield clearly positive reactions with the 3 high-titre rheumatoidsera. In this manner the test interference is immediately recognized anda false-positive result is avoided. The MAB “2” is most suited toindicate rheumatoid factor interference.

1. Solid phase with at least one defined test area for the detection ofan analyte in a sample, wherein it additionally comprises at least onedefined control area for the detection of interferences.
 2. Solid phaseas claimed in claim 1, wherein it comprises several test areas for thesimultaneous detection of several analytes in a sample.
 3. Solid phaseas claimed in one of the previous claims, wherein it comprises at leastone control area to detect interferences which are caused by anon-analyte-specific binding of components of the detection medium tothe test area.
 4. Solid phase as claimed in one of the previous claims,wherein it comprises at least one control area to detect interferenceswhich are caused by a non-analyte specific binding of components of thedetection medium to the solid phase receptor on a test area.
 5. Solidphase as claimed in claim 3 or 4, wherein it comprises at least onecontrol area to detect interferences which are caused by a non-analytespecific binding of components of the detection medium to immobilizedantibodies, antibody fragments, antigens or/and nucleic acids on a testarea.
 6. Solid phase as claimed in claim 4 or 5, wherein the controlarea contains a modified solid phase receptor which differs from a solidphase receptor on a test area by the absence of an analyte-specificbinding site.
 7. Solid phase as claimed in claim 4, 5 or 6, wherein itcomprises at least one control area to detect interferences byrheumatoid factors.
 8. Solid phase as claimed in claim 4, 5 or 6,wherein it comprises at least one control area to detect interferencesby foreign-species-specific antibodies.
 9. Solid phase as claimed in oneof the previous claims, wherein it comprises at least one control areato detect interferences which are caused by unspecific binding ofcomponents of the detection medium to non-analyte-specific substances onthe solid phase support.
 10. Solid phase as claimed in claim 9, whereinit comprises at least one test area containing streptavidin and at leastone control area to detect interferences by non-analyte-specificstreptavidin-binding components of the detection medium.
 11. Solid phaseas claimed in claim 3, wherein it comprises at least one control area todetect the total IgE content.
 12. Solid phase as claimed in one of theprevious claims, wherein it additionally comprises at least one positivereference area containing the analyte to be determined.
 13. Solid phaseas claimed in one of the previous claims, wherein the test areas andcontrol areas each have a diameter of 10 μm to 1 cm.
 14. Use of a solidphase as claimed in one of the claims 1 to 13 to detect an analyte in asample.
 15. Use as claimed in claim 14, wherein the solid phasecomprises several test areas for the simultaneous detection of severalanalytes.
 16. Use as claimed in claims 14 or 15 in an immunoassay. 17.Use as claimed in claims 14 or 15 in a nucleic acid hybridization assay.18. Method for the detection of an analyte using a solid phase with atleast one defined test area which additionally comprises at least onedefined control area to detect interfering reactions.
 19. Method asclaimed in claim 18, wherein control areas are used to quantitativelycorrect for interferences.
 20. Use of defined control areas for thesimultaneous detection and optionally for the quantitative correction ofinterferences in a method for the detection of an analyte.